基于分子修饰的黑米花青素对肠道益生菌的增殖作用及构效关系研究

Prebiotics plays an indispensable role on the normal function of the intestinal microecology. Black rice anthocyanins are prebiotics, however, their proliferation effect on intestinal probiotics and the structure-activity relationship has not yet been elucidated. In previous study, we discovered that crude black rice anthocyanins could improve antioxidant capacity in vivo and promote the proliferation of Bifidobacterium and Lactobacillus in vitro as well. In addition, cyanidin-3-glucoside (C3G), paeoniflorin-3-rutanoside (P3R) and other four components were isolated from black rice, and their antioxidant and prebiotic activity in vitro were elucidated. Based on these, in this project, organic acids as acyl donors, C3G and P3R, which are used as starting molecules, will be modified by enzymatic acylation, and the reaction conditions and mechanism would be studied. On the basis of toxicological evaluation, the structural characteristics, physical and chemical properties and probiotics proliferation of the modified target products would be studied. And the structure-activity relationship, which promotes the proliferation of intestinal probiotics, would be analyzed. Furthermore, the effect of modified products in vitro on the mRNA expression of gene, which controls the major glycosidase enzymes secretied by probiotic cells will be analyzed. The effect of anthocyanins on the diversity of intestinal microflora, intestinal contents, the sIgA content of Intestinal mucosal and the fixed value of microbial in vivo will be discussed as well. The effect of black rice anthocyanins on probiotics proliferation would be studied on global, cellular and molecular levels, respectively.

益生元对肠道微生态正常功能的发挥起着举足轻重的作用。黑米花青素作为益生元的一种，对肠道益生菌的增殖作用及构效关系尚未阐明。在前期研究中，我们发现黑米花青素粗提物具有提高体内外抗氧化作用，促进双歧杆菌及乳酸菌增殖作用；分离出矢车菊素-3-葡萄糖苷（C3G）及芍药素-3-芸香糖苷（P3R）等六种组分，阐明了其体外抗氧化及益生元活性。本项目拟以C3G及P3R为起始分子，有机酸为酰基供体，采用酶法酰基化修饰，进行分子改造，研究反应条件及机理。在毒理学评价的基础上，进行结构特征、理化性质及益生菌的增殖作用研究，解析其促进肠道益生菌增殖的构效关系。在体外，分析其对益生菌的代谢产物及细胞分泌主要糖苷酶基因mRNA表达的影响；在体内，分析花青素对肠道菌群多样性、肠道内容物、肠粘膜sIgA含量、微生物定值力的影响。从整体、细胞及分子水平三个层次，研究黑米花青素对益生菌的增殖作用机理。

本课题以黑米花青素为研究对象，分离出矢车菊-3-葡萄糖苷，以其为起始分子，采用分子修饰，获得花青素酰基产物；通过液质联用和核磁共振等技术手段，分析产物的结构，并且探讨了反应动力学；通过吸光值、热降解动力学、色差等分析，探讨温度、pH值、过氧化氢、抗坏血酸和金属离子对修饰产物稳定性的影响；通过急性、亚急性和亚慢性毒性试验，对产物的毒理性进行评价。测定小鼠进食产物后死亡率、体重、进食饮水量、病理组织学、脏器指数及生化指标，建立体内安全性评价模型；研究了修饰产物对益生菌的体外增殖作用；通过模拟人体胃肠道环境，研究了产物的抗消化性；以体外厌氧培养的方式，通过测定益生菌生长情况，研究花青素修饰产物的益生元活性；通过对代谢过程中菌落数、pH值、生长速率和短链脂肪酸的测定，评价花青素修饰产物对肠道菌群增殖的影响。采用气质，分析产物对人体肠道菌群代谢物的影响；通过体内试验，分析小鼠进食产物后的体重、肠道内容物pH、肠道组织形态、免疫细胞活性、肠道消化酶活性（蛋白酶、淀粉酶、脂肪酶）以及微生物菌群多样性等指标，对产物的肠道免疫活性进行评价；以单核细胞增生李斯特菌、金黄色葡萄球菌、肠炎沙门氏菌和副溶血弧菌为作用对象，研究了花青素酰基产物对病原菌的抑制作用。结果表明，经过酰基化后的抗消化能力明显提高；能够有效地抑制痢疾志贺氏菌、小肠结肠炎耶尔森菌、肠炎沙门氏菌和伤寒沙门氏菌的生长，提高了益生菌如乳杆菌的生长，发挥调节肠道菌群结构的作用；对小鼠肠道内的胰蛋白酶、淀粉酶的活力显著提高，对脂肪酶的活力有显著降低。黑米花色苷酰基化产物是安全无毒的，对小鼠的生长无剂量毒性影响。并且，修饰产物的脂溶性得到显著改善，且抵抗温度、pH值变化、氧化剂和部分金属离子的能力更强。.本项目的研究结果将为更好地了解黑米花青素修饰产物的益生元活性及作用机制，为其产业化开发提供一定的技术支撑和理论基础。